DNA helicases are motor proteins that couple the energy generated through nucleotide binding and hydrolysis to DNA unwinding and translocation. Interestingly, although the monomeric form of Rep is incapable of initiating DNA unwinding in vitro, preliminary evidence suggests that Rep monomers may be capable of translocating along single-stranded (ss) DNA. This translocation ability likely plays a role in the unwinding mechanism of the active oligomeric form of the enzyme. The focus of this proposal is to characterize the kinetic mechanism for AlP binding and hydrolysis by Rep monomers bound to ss DNA and to determine whether Rep monomers are, indeed, capable of translocation along ss DNA. The specific aims for this proposal are (1) to investigate the mechanism of monomeric Rep's association with and dissociation from ss DNA; (2) to investigate the kinetic mechanism for nucleotide binding and hydrolysis by Rep monomers bound to ss DNA; (3) to determine if the Rep monomer can translocate along ss DNA and if this translocation is directionally biased; and (4) to investigate the processivity of Rep monomer translocation along ss DNA and determine the thermodynamic coupling efficiency of ATP hydrolysis to Rep monomer translocation.